Cystic fibrosis (CF) is the most common life-shortening disease in Caucasians (12,63). Although severity of the disease is variable, a large proportion of affected individuals develop obstructive lung disease with progressive loss of pulmonary function. Mucus plugging, poor ciliary function, chronic inflammation and infection of the airways are common manifestations of the disease (63). Recently it was observed that airway epithelia from CF patients exhibit abnormal regeneration which is exacerbated by infection and inflammation (28). The role of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) in this process is poorly understood. Preliminary experiments presented in this application show that CFTR function is required for efficient airway epithelial cell migration and wound repair. Blocking channel activity or silencing expression by RNA interference produced a significant delay in wound healing by impeding lamellipodia protrusion and reducing the rate of cell migration into the wound. Moreover, our most recent preliminary data using bronchial epithelial cells obtained from CF patients expressing the most common (?F508) mutation also exhibited significantly delayed wound healing compared to cells from normal subjects. Thus the first objective of this proposal is to understand the mechanism by which CFTR participates in airway epithelial cell migration and restitution. The hypothesis suggested by our preliminary experiments is that CFTR promotes migration through regulation of extracellular pH, particularly at the leading edge of the cell and that reduced channel activity enhances adhesion and decreases lamellipodia protrusion and the rate of cell migration. The second objective is to investigate the molecular mechanisms responsible for Cl- and HCO3- dependency of cell migration mediated by the putative functional coupling of CFTR and anion exchangers in primary human bronchial epithelial cells. We propose that a Cl-HCO3 exchanger functions in parallel with CFTR to mediate Cl- uptake in exchange for HCO3 efflux into the extracellular matrix (ECM). CFTR provides a critical pathway for Cl- recycling which sustains the activity of the Cl-HCO3 exchanger so that buffering capacity within the ECM is maintained. To address these hypotheses, we plan to measure changes in extracellular pH (pHe) at the membrane-ECM interface using a ratio imaging approach and to apply self-referencing ion-selective microelectrode technology to study the role of CFTR in regulating pHe within the membrane-ECM microenvironment. We will also determine the molecular identity of the anion exchanger and examine its role in regulation of ECM pH. Understanding the cellular mechanisms involved in the contribution of CFTR to airway epithelial cell repair and restitution may have significant impact on the development of new pharmacotherapies that could potentially reduce the exacerbating effects of infection, chronic inflammation and remodeling that contribute to the progressive loss of pulmonary function observed in CF. PUBLIC HEALTH RELEVANCE: Despite dramatic advances in our knowledge of the genetic and molecular basis for CF there are still significant gaps in our understanding of the pathogenic events involved in pulmonary dysfunction. Our preliminary findings highlight a previously uncharacterized role for CFTR in the process of wound healing that could enhance our understanding of CF pathophysiology. Insights gained from the proposed studies may lead to identification of new drug targets or therapeutic strategies to limit the progressive deterioration of lung function.